tion, ring-opening polymerization (ROP), click reaction and atom transfer radical polymerization (ATRP).
One identified polymer construct (P4) was able to form stabilized unimolecular micelles in an aqueous
solution with a diameter of approximately 68 nm and showed the highest fluorescence quantum yield of
0.55, which is on a par with that of the small organic molecular fluorophore standard, quinine sulfate. The
potential of P4 for simultaneous cell imaging and drug delivery was further evaluated in vitro, which
confirmed efficient cellular uptake and cytotoxicity in HeLa cells. This study thus presents the first
example of PF-backboned bb copolymers with alternating heterobrushes for cancer theranostics.
Introduction
Polymers with advanced topological structures such as star-shaped polymers,1–3 hyperbranched polymers,4–9 cyclicpolymers,10–12 and bottlebrush (bb) polymers13–25 show uniqueproperties relative to their traditionally linear analogues with anidentical molecular weight (MW), such as a smaller hydrodyn-amic radius, multi-valent polymer surface toward more func-tionalities, no or lower chain entanglement, and capability toform unimolecular micelles with greater stability; thus thedesign and precise synthesis of these polymers for variouspotential applications have been a hot subject of research for
several decades and have drawn increasing attention in recentyears. Among these structures, bb polymers with densely graftedpolymer brushes represent an intriguing advanced macro-molecular architecture due to the greater stability and drugloading capacity of their self-assembled micelles than those oftheir linear counterparts toward minimized side effects andenhanced therapeutic efficiency for drug delivery.14,16,17,25–27
Compared to the extensive and intensive investigations on bbcopolymers composed of homogeneous polymer brushes,10–12
the preparation of bb copolymers with heterogeneous polymergrafts17 remains relatively unexplored likely due to the syntheticchallenge. bb copolymers with hetero-polymer brushes can self-assemble into more complex nanoassemblies including multi-compartment micelles and Janus-type cylinders with tunablefunctions and properties in selective solvents due to the uniquephase separation of the pendant side chains with different pro-perties. Therefore this self-assembly process of polymer speciesintegrating two or more hetero-polymer brushes provides newinsights into the properties of macromolecules with advancedtopologies as well as their potential for various applications.17
Together with the elegant adoption of functional polymers, e.g.,conjugated polymers as the backbone, the resulting bb copoly-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c8py01221k‡These authors contributed equally to this paper.
aState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous
Metal Chemistry and Resources Utilization of Gansu Province, and College of
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000,
China. E-mail: [email protected] of Pharmaceutics, School of Pharmacy, Hubei University of Chinese
mers integrate simultaneously the abilities of cell-imaging fordiagnosis and drug release for therapy.13,15,20,22
Polyfluorene (PF) is one of the most investigated conjugatedpolymers with superior fluorescence properties and both chemi-cal and thermal stabilities.28–47 More importantly, the C9 posi-tion of PF could be easily modified with a variety of functionalgroups, thus offering endless possibilities for chemistry decora-tions and biomedical applications.35,37,42–47 Liu et al. syn-thesized a conjugated polyelectrolyte with pendant oligopeptidebrushes using a facile strategy of click chemistry.22 Wang et al.prepared a series of amphiphilic conjugated bb copolymerswith the backbone of fluorescent poly(fluorene-alt-(4,7-bis(hexylthien)-2,1,3-benzothiadiazole)) (PFTB) grafted by the sidechains of amphiphilic poly(ε-caprolactone)-block-poly(oligo(ethylene glycol)methyl ether methacrylate) (PCL-b-POEGMA)copolymers.15 However, compared to the well-developedPF-backboned bb copolymers with homobrushes,13,15,20–24 theincorporation of heterobrushes remains unexplored likely dueto the synthetic challenges. For this purpose, we reported in thisstudy the synthesis of a series of well-defined amphiphilic con-jugated bb copolymers, PF-((g-PCL-OOCCH3)-alt-(g-POEGMA))(Scheme 1). The resulting amphiphilic conjugated bb copoly-mers are composed of not only a PF backbone with strong bluefluorescence for cell imaging, but also alternating POEGMA/PCLbrushes capable of preventing the conjugated backbone fromaggregation toward increased fluorescence quantum yields ofPF. The potential of this formulation for simultaneous cellimaging and drug delivery was further evaluated in vitro by fluo-rescence microscopy and in vitro cytotoxicity study.
Results and discussionSynthesis and characterization of PF-((g-PCL-OOCCH3)-alt-(g-POEGMA)) copolymers
Well-defined amphiphilic conjugated bb copolymer PF-((g-PCL-OOCCH3)-alt-(g-POEGMA)) was prepared in four steps
including (a) synthesis of the PF backbone with alternatingazide and hydroxyl functions in the termini of the pendantgrafts, PF-((g-N3)-alt-(g-OH)) (Scheme 2a), (b) synthesis ofalkyne-PCL-OH by Sn(Oct)2-catalyzed ROP of ε-CL using propy-nol as an initiator, and subsequent end capping of the reactivehydroxyl group to unreactive acetate by esterification(Scheme 2b), (c) conjugation of PCL grafts to the PF backboneby click coupling between PF-((g-N3)-alt-(g-OH)) and alkyne-PCL-OOCCH3 with an azide and alkyne molar feed ratio of1 : 0.85 (Scheme 3), and (d) introduction of ATRP initiatingsites into the above polymers by an esterification reaction withexcess 2-bromoisobutyryl bromide, and production of targetamphiphilic conjugated bb copolymers PF-((g-PCL-OOCCH3)-alt-(g-POEGMA)) by ATRP of OEGMA using a PF-((g-PCL-OOCCH3)-alt-(g-Br)) multimacroinitiator (Scheme 3). TheMW, degree of polymerization (DP) and polydispersity (ÐM) ofall the synthesized polymers are summarized in Table 1. Notethat the MWs of the synthesized polymers determined by 1HNMR analyses are estimations rather than accurate calcu-lations as detailed in Table 1.
Fig. S4† presents the typical 1H NMR spectra of alkynyl-PCL30-OH and alkyne-PCL30-OOCCH3. The DP of CL (Fig. S4a†)was determined to be approximately 30 according to our pre-vious NMR analyses.2,31 The terminal hydroxyl group of PCLwas next converted to acetate by a reaction with anhydrousacetic acid and oxalyl chloride to avoid its transformation toATRP initiating sites for the generation of POEGMA brushes inthe fourth step of polymer synthesis mentioned above. The fullend-capping of the hydroxyl termini by acetate functions wasconfirmed by the appearance of a new methyl signal (peak g)
Scheme 1 Schematic representation of the synthesis, drug loading,cellular uptake, and intracellular drug release of the amphiphilic conju-gated bb copolymers with alternating PCL/POEGMA grafts.
Scheme 2 Synthesis of monomers 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(6’-azidohexyl)fluorine (1) and 2,7-diiodo-9,9-bis(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-fluorene (2), and polymersPF-((g-N3)-alt-(g-OH)) (a) and alkynyl-PCL-OOCCH3 (b).
at 2.04 ppm and a ratio of 2/3 of the integrated intensity ofpeak a (the methylene protons adjacent to alkynyl) to peak g(the protons of the methyl termini) (Fig. S4b†). The unimodalSEC elution peak with narrow distribution recorded for alkyne-PCL30-OOCCH3 demonstrates a well-controlled ROP process(Fig. 2a). The successful synthesis of the well-defined parentpolymer of PF with alternating azide and hydroxyl functions inthe termini of the pendant grafts, PF-((g-N3)-alt-(g-OH)), throughthe Suzuki coupling reaction between monomer 1 (Fig. S1†) andmonomer 2 (Fig. S2†), was confirmed by 1H NMR andSEC-MALLS analyses (Fig. S3† and Fig. 2a). The resulting PF-((g-N3)-alt-(g-OH)) also indicates a unimodal SEC elution peak
with narrow distribution, and the DP of PF was determined tobe ∼13 based on the MW determined by SEC-MALLS.
The hydrophobic PCL grafts, alkyne-PCL30-OOCCH3, weresubsequently conjugated to the parent PF, PF-((g-N3)-alt-(g-OH)), by a copper(I)-catalyzed azide–alkyne cycloaddition(CuAAC) click reaction to generate the conjugated bb copoly-mer, PF13-((g-PCL30-OOCCH3)-alt-(g-OH)). Note that the azidegroup was used in slight excess of the alkyne function with amolar feed ratio of 1 : 0.85 for simplified purification, takingadvantage of the high efficiency of the CuAAC click-grafting.Successful polymer synthesis was confirmed by the appearanceof all the characteristic signals of both PF and PCL moieties(Fig. 1a), a clear change of the signal at 3.15 ppm attributed tothe methylene protons adjacent to azide in the 1H NMR spec-trum of PF-((g-N3)-alt-(g-OH)) (Fig. S3†) to 4.20 ppm in the 1HNMR spectrum of PF13-((g-PCL30-OOCCH3)-alt-(g-OH)) (Fig. 1a)after click coupling as well as a notable shift of its SEC elutiontrace toward a higher MW relative to the parent polymer of PF(Fig. 2a). Moreover, the high purity of the synthesized PF13-((g-PCL30-OOCCH3)-alt-(g-OH)) was corroborated by a completeshift of the resonance signal at 4.67 ppm attributed to themethylene protons adjacent to alkynyl in the 1H NMR spec-trum of alkyne-PCL30-OOCCH3 (Fig. S4a†) to 5.14 ppm in the1H NMR spectrum of PF13-((g-PCL30-OOCCH3)-alt-(g-OH))(Fig. 1a) after click grafting as well as its narrowly distributedSEC elution peak (Fig. 2a).
Next, the pendant hydroxyl groups of PF13-((g-PCL30-OOCCH3)-alt-(g-OH)) were converted to ATRP initiators forgeneration of POEGMA brushes by a grafting-from approach.The successful introduction of ATRP initiating units was con-firmed by a respective shift of the resonance signals at 3.64and 3.49 ppm attributed to the protons of the methylene adja-cent to the hydroxyl in the 1H NMR spectrum of PF13-((g-PCL30-OOCCH3)-alt-(g-OH)) (Fig. 1a) to 4.23 and 3.64 ppm(Fig. 1b) and the appearance of a new resonance signal (peakt) attributed to the methyl of the initiating units at 1.89 ppmin the 1H NMR spectrum of PF13-((g-PCL30-OOCCH3)-alt-(g-Br))(Fig. 1b) after the reaction. Moreover, the ratio of the inte-grated intensity of peak t and peak (l + g) assigned to theprotons of the methylene adjacent to 1,2,3-triazole andhydroxyl of PF13-((g-PCL30-OOCCH3)-alt-(g-OH)) was calculated
Scheme 3 Synthesis of bb copolymers PF-((g-PCL-OOCCH3)-alt-(g-POEGMA)) via an integrated approach of “graft to” and “graft from”.
Table 1 MW, ÐM, and DP of all the synthesized polymers
a The DP of the backbone PF13-((g-N3)-alt-(g-OH)) was calculated from SEC-MALLS results; the DP of PCL and POEGMA brushes was calculatedfrom 1H NMR results based on the DP of N3-PF-OH. b Mn, NMR was estimated from 1H NMR results. c Mn, SEC-MALLS was calculated fromSEC-MALLS results.
to be 3/2, indicating complete decoration of the ATRP initiat-ing sites.
The ATRP kinetic study using a PF13-((g-PCL30-OOCCH3)-alt-(g-Br)) multimacroinitiator was carried out at various polymer-
ization periods with a target DP of 100 for each initiating site.The SEC elution traces of the prepared four conjugated amphi-philic bb copolymers, PF-((g-PCL-OOCCH3)-alt-(g-POEGMA)),show a detectable shift toward the higher MW with thepolymerization time (Fig. 2b). During the evaluated polymeriz-ation process, the living characteristics were reflected by thepseudo-first-order kinetics and an almost constant ÐM around1.60 (Fig. 2c & d). The acquired relatively broad ÐM relative tothose of the linear polymers is reasonable given the more com-plicated polymer structure of bb copolymers and similar valuesreported in previous studies.4,6,16,31,35 The DP of POEGMAbrushes was calculated by comparing the integrated intensityof peak l at 3.38 ppm attributed to the protons of the methoxyltermini of POEGMA brushes and peak a assigned to thecharacteristic signal of PCL grafts (Fig. 1c).
Size and morphology of self-assembled micelles
The size of polymeric micelles is a critical factor that exerts asignificant effect on the performance of micelle drug carriers.To achieve efficiently passive tumor targeting, the polymermicelles should have a relatively small size (<100 nm), whichcan guarantee a lower level of nonspecific uptake by theReticuloendothelial System (RES), minimal renal excretion,and promotion of the enhanced permeability and retention(EPR) effect.48,49 The ability of four amphiphilic conjugated bbcopolymers PF13-((g-PCL30-OOCCH3)-alt-(g-POEGMA)) (P1–P4)
Fig. 1 1H NMR spectra of (a) PF13-((g-PCL30-OOCCH3)-alt-(g-OH)), (b) PF13-((g-PCL30-OOCCH3)-alt-(g-Br)), and (c) PF13-((g-PCL30-OOCCH3)-alt-(g-POEGMA38)).
Fig. 2 SEC elution traces (dRI signals) of (a) PF13-((g-N3)-alt-(g-OH)),alkyne-PCL30-OOCCH3, and PF13-((g-PCL30-OOCCH3)-alt-(g-OH)),(b–d) ATRP kinetics study of the conjugated bb copolymers using thePF13-((g-PCL30-OOCCH3)-alt-(g-Br)) multimacroinitiator with a targetDP of 100 for each initiating site.
to form unimolecular micelles was further evaluated usingN,N′-dimethylformamide (DMF) as the medium that is a goodsolvent for all the moieties of the synthesized bb copolymersincluding the PF backbone, alternating brushes of PCL andPOEGMA. Given the non-occurrence of self-assembly of theresulting bb copolymers in DMF, the mean size determined bydynamic light scattering (DLS) in DMF at a polymer concen-tration of 0.1 mg ml−1 reveals an actual dimension reached bya free polymer chain. On the other hand, self-assembly of theresulting bb copolymers in water takes place due to theiramphiphilicity; therefore the average size determined by DLSin water at an identical polymer concentration of 0.1 mg ml−1
indicates a statistical diameter of the nano-objects self-assembled by the polymers. A close value of the two sizes sup-ports the formation of unimolecular micelles consisting of asingle polymer chain because association of polymer chains inan aqueous phase leads to a significantly larger size of theformed aggregates relative to that of a free polymer chaindetermined in DMF.
The mean size shows an increasing trend following theorder of P4 > P3 > P2 > P1 in DMF (Table 2 and Fig. S5†), whichindicates that the differences in the sizes of their self-assem-blies are substantially dependent on the POEGMA brushes andthe longer chain length of POEGMA brushes results in a largerdimension of a bb polymer chain because all the synthesizedfour amphiphilic conjugated bb copolymers have the same PFbackbone and pendant hydrophobic PCL brushes but differentchain lengths of hydrophilic POEGMA grafts. More impor-tantly, it is interesting to notice that P4 exhibits quite similarsizes in DMF and water (64.30 vs. 67.63 nm) at an identicalpolymer concentration of 0.1 mg mL−1 (Table 2), supportingits formation of unimolecular micelles in an aqueous solution.However, the other three polymers, P1, P2, and P3, show a sig-nificantly greater size in water than those recorded in DMFlikely due to the association of polymer chains with micellestructures for particulate stabilization. Therefore the trend ofsize recorded in water is quite different from the tendencyobserved in DMF. The diversities observed in the mean sizedetermined in water are relevant to the dimension of a freepolymer chain and the different aggregation numbers of theformed self-assemblies. The sizes of the self-assembledmicelles are also affected by the parameters such as the mole-cular weight, polymer composition, concentration of solutionand so on.50 Such unique self-assembly behaviors of P4 shouldbe attributed to its longest POEGMA brushes among all thefour bb copolymers. The strongest steric hindrance resultingfrom the longest POEGMA brushes of P4 provides sufficient
stability for its formation of unimolecular micelles rather thanaggregates associated by the free polymer chains in water.
Transmission electron microscopy (TEM) was further per-formed to provide morphological insight into the self-assem-blies formed by the amphiphilic conjugated bb copolymers(Fig. 3 & Fig. S6†), which reveals the formation of the well-dis-persed micelles with a regular spherical shape for all the fourbb copolymers. The average diameter of micelles self-assembled by P3 and P4 at a polymer concentration of 0.5 mgmL−1 was estimated to be approximately 42 and 28 nm,respectively, in water from the TEM images (Fig. 3c & d, thecorresponding mean sizes determined by DLS are presented inFig. 3a & b). To validate the unique formation of unimolecularmicelles by P4, TEM visualization was carried out in DMF aswell. As expected, the average size of P3 micelles in water isclearly larger than that observed in DMF (∼21 nm fromFig. 3e). In contrast, P4 micelles exhibit a mean size of ∼30 nmin DMF (Fig. 3f) that is almost identical to the value deter-mined in water. The results agree well with the DLS data andstrongly support the greater stability of P4 micelles relative tothe other three micelle constructs due to the longest chainlength of POEGMA brushes of P4.
Various factors in the physiological systems, such as ionicstrength, dilution with bloodstream and proteins, also exert a
Table 2 Summary of the mean sizes and polydispersity indexes (PDIs)of the synthesized four amphiphilic bb copolymers, P1–P4, determinedby DLS in water and DMF at a polymer concentration of 0.1 mg mL−1
Dh (nm)/PDI P1 P2 P3 P4
Water 97.75/0.350 77.39/0.218 81.67/0.217 67.63/0.208DMF 33.61/0.294 37.45/0.222 43.59/0.277 64.30/0.324
Fig. 3 Size distributions of (a) P3 and (b) P4 micelles in water, TEMimages of (c & e) P3 and (d & f) P4 micelles in (c & d) water and (e & f)DMF at a polymer concentration of 0.5 mg mL−1.
significant effect on the structural integrity of the self-assembled micelles.15,51 To evaluate the stability of micellesformed by P3 and P4 under salt and diluted conditions, theaverage diameter at various concentrations in different mediawas determined by DLS (Fig. S7a† & Fig. 4a). The average sizeof P4 micelles remains almost identical in the tested polymerconcentrations ranging from 0.1 to 2.0 mg mL−1 in both waterand PBS (Fig. 4a), confirming the apparent stability of P4micelles irrespective of salt addition and dilution due to theformation of unimolecular micelles with enhanced stability.On the other hand, P3 micelles showed consistently slightlysmaller average diameters in water than those recorded in PBSlikely due to the salt effect. Both values remain constant in therange of polymer concentrations from 0.05 to 1.0 mg mL−1,also implying the stability of micelles under the physiologicalsalt and diluted conditions (Fig. S7a†).
In vitro drug loading and drug release studies
To investigate the potential performance of the amphiphilicconjugated bb copolymers for drug delivery applications,in vitro drug loading and drug release studies were performedusing doxorubicin (DOX) as the model drug. P3 and P4micelles with enhanced stability were chosen to encapsulateDOX via a classical dialysis method to produce two theranosticmicelles of DOX@P3 and DOX@P4.
After encapsulation of DOX within the hydrophobic core ofthe micelles, the average diameters of both drug-loadedmicelles showed a slight increase (Fig. S7c† & Fig. 4c) relativeto those of the blank micelles (Fig. S7b† & Fig. 4b) in PBS and(Fig. S7d† & Fig. 4d) in the presence of 10% fetal bovine serum(FBS), which demonstrates that drug encapsulation increasessomewhat the micelle size. It is important to note that theDOX@P4 micelles show a better symmetrical size distributionand a smaller PDI relative to the DOX@P3 micelles, probablyimplying the greater stability of unimolecular micelles of P4
relative to the micelles self-assembled by P3. Such greaterstability also contributed to the higher drug loading capacityof P4 micelles, leading to slightly larger DLC and EE ofDOX@P4 micelles (4.2% and 46%) relative to DOX@P3 ana-logues (3.8% and 42%).31
The in vitro DOX release profiles of the two theranosticmicelles were investigated at 37 °C under the physiologicalconditions (PBS, pH 7.4, 150 mM) simulating the typical extra-cellular pH, and in an acidic medium (SSC, pH 5.0 150 mM)mimicking the tumor intracellular pH, respectively. As shownin Fig. 5, DOX release of DOX@P4 micelles in the acidicmedium of pH 5.0 was consistently faster than that recordedunder the physiological conditions of pH 7.4, i.e., 60% DOXrelease at pH 5.0 vs. 40% DOX release at pH 7.4 in 96 h(Fig. 5), which is primarily attributed to the promoted protona-tion of the glycosidic amine toward increased solubility ofDOX in an acidic medium.31 Based on the current results,both release profiles gradually levelled off after 48 h, approach-ing the “zero-order” kinetics.52 Therefore a greater cumulativedrug release could be achieved for a long-term drug release.
An identical trend of in vitro DOX release was observed forDOX@P3 micelles, which mediated a slightly higher cumulat-ive drug release at both pH values, i.e., 65% at pH 5.0 and 46%at pH 7.4 in 72 h (Fig. S8†), than DOX@P4 formulations, likelyrelevant to the differences in micelle stabilities.
Photophysical properties
The optical properties include the absorption and emissionspectra, as well as the fluorescence quantum yields of conju-gated polymers. It has been repeatedly highlighted that aggre-gation of conjugated polymers leads to significantly reducedfluorescence quantum yields. In this study, the phase-separ-ation of the hetero-polymer brushes can prevent the PF back-bone from aggregation and contribute to the increased fluo-rescence quantum yields of the synthesized four amphiphilicconjugated bb copolymers. To clarify the structure–propertyrelationship, the photophysical properties of the synthesizedfour amphiphilic conjugated bb copolymers, P1–P4, were inves-tigated in water and DMF at room temperature. All the poly-
Fig. 4 Average sizes of P4 at various concentrations in water and DMFdetermined by DLS, size distributions of (b) P4 in PBS, (c) DOX@P4 in PBSand (d) DOX@P4 in the presence of 10% FBS at a polymer concentrationof 1 mg mL−1.
Fig. 5 In vitro drug release profiles of DOX@P4 micelles at different pHvalues of 7.4 and 5.0 at 37 °C.
mers showed similar photophysical properties in both solvents(Fig. 6a, Fig. S9 & S10a†) with the typical absorption peak at330–430 nm and the characteristic emission peak at400–520 nm from the conjugated PF backbone when excited at365 nm. The photophysical properties of DOX@P3 andDOX@P4 micelles were also investigated and compared tothose of free DOX in water (Fig. 6b, Fig. S10b & S11†). Therewas a notable presence of the characteristic DOX absorption inthe UV-Vis spectrum, but the emission spectrum of DOX wasundetectable likely due to the excitation wavelength of 365 nmused for the PF moiety.
The fluorescence quantum yields of P1–P4 were calculatedusing quinine sulfate in 0.5 mol L−1 H2SO4 (ΦF = 0.55) as thestandard. The emission spectrum was recorded with an exci-tation wavelength of 365 nm. By comparing the integratedfluorescence spectrum of the polymers with that of quininesulfate based on the correction of the refractive index differ-ences in water and DMF, the fluorescence quantum yields ofall the four bb copolymers in DMF were determined to besimilar values (Table 3), but a significant increase of the fluo-rescence quantum yields from 0.35 to 0.55 in water wasnoticed following the order of P1 < P2 < P3 < P4 with increasingchain length of the hydrophilic POEGMA brushes from 12 to38, which possibly implies that the longer POEGMA graftsprovide better prevention of the PF backbone from aggregationand subsequent fluorescence quenching toward greater fluo-rescence properties. The photostability is reflected by the fluo-rescence quantum yields of polymers in water in this study.
Based on the results, P4 micelles with the greater photo-stability than the other three micelles of P1–P3 were identifiedas the best polymer construct. The results agree well with the
best stability of P4 micelles. Most importantly, the fluorescencequantum yields of the unimolecular P4 micelles in water areeven as high as that of the quinine sulfate standard that is asmall organic molecular fluorophore, confirming the greatpotential of P4 micelles as a fluorescent probe for cellularimaging.
Cellular imaging and in vitro cytotoxicity
To evaluate the potential of DOX@P3 and DOX@P4 micelles incellular imaging and drug delivery, the cellular uptake ofmicelles was observed by fluorescence microscopy. Note thatHeLa cells were stained with acridine orange (AO) dye to dis-tinguish the nuclei (Fig. 7b & Fig. S12,† indicated usingarrows) from the cytoplasm stained in green. Taking theimages of HeLa cells incubated with DOX@P4 micelles as anexample, the obvious blue fluorescence from the PF segmentand the strong red fluorescence from DOX throughout the cel-lular cytoplasm and the perinuclear region (Fig. 7a & c), as wellas the overlay image (Fig. 7d) confirm the efficient endocytosisof DOX@P4 micelles into the cytoplasm of HeLa cells andtheir excellent abilities for cellular imaging and drug delivery.DOX@P3 micelles showed similar cellular uptake behaviors(Fig. S12†).
Finally, the in vitro cytotoxicity of DOX@P3 and DOX@P4micelles as well as the blank micelles was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium (MTS) cell viability assay againstHeLa cells (Fig. 8, Fig. S13 & S14†). The blank micelles of P3and P4 are almost non-toxic to the cells, with a cell viability of80% even at the highest tested polymer concentration of2.6 mg mL−1 (Fig. S13†). The half maximal inhibitory concen-tration (IC50) of free DOX, DOX@P3 and DOX@P4 micelles is2.33 (2.27, 2.40) μg mL−1 (Fig. S15†), 89.44 (87.0, 101.5)μg mL−1 (Fig. S14†) and 87.60 (86.02, 110.3) μg mL−1 (Fig. 8),respectively. The DOX-loaded micelles exhibited significantcytotoxicity to HeLa cells, but had a less cytotoxic activity thanfree DOX likely due to the slower internalization mechanism(endocytosis vs. direct membrane permeation) and release
Fig. 6 UV-Vis absorption and fluorescence emission spectra of (a) P4 inwater and DMF and (b) DOX@P4 in water.
Table 3 Summary of the photophysical properties of the four amphi-philic bb copolymers PF13-((g-PCL30-OOCCH3)-alt-(g-POEGMA)) inwater and DMF
Sample Solvent λmax, absa (nm) λmax, em
b (nm) Φc
P1 Water 386 423 0.35P1 DMF 393 419 0.75P2 Water 387 421 0.40P2 DMF 392 422 0.80P3 Water 388 423 0.51P3 DMF 391 422 0.73P4 Water 386 420 0.55P4 DMF 393 420 0.82
a The absorbance λmax was determined from UV-Vis spectra. b Theemission λmax was determined from fluorescence spectra with exci-tation at 365 nm. c The fluorescence quantum yields of the amphiphi-lic bb copolymers in water and DMF were measured using quininesulfate in 0.5 mol L−1 H2SO4 (ΦF = 0.55) as the standard.
kinetics of the free drug from the micelles.31 The quite similarIC50 values of DOX@P3 and DOX@P4 micelles are attributed tothe similar cumulative DOX release at pH 5.0.
Conclusions
In summary, we synthesized a series of amphiphilic conju-gated bb copolymers with POEGMA/PCL heterografts by anintegration of “grafting to” and “grafting from” techniquesfrom the backbone of a PF-based parent polymer. P4 wasidentified as the optimal polymer construct capable of
forming stabilized unimolecular micelles in an aqueous solu-tion with the highest fluorescence quantum yield of 0.55,which is on a par with that of the small organic molecularfluorophore standard, quinine sulfate. The potential of P4 forsimultaneous cell imaging and drug delivery was further evalu-ated in vitro, which confirmed efficient cellular uptake andcytotoxicity in HeLa cells. The current bb copolymers can befurther modified with various strategies to develop multifunc-tional nanocarriers toward enhanced anticancer drug delivery,including (a) active targeting achieved by conversion of thebromo termini of POEGMA brushes to azide functions forfurther click coupling with various targeting ligands (e.g. anti-body, peptide and small molecule ligand) and (b) efficientintracellular drug release realized by the introduction ofdifferent stimuli-responsive polymers as the pendant polymerbrushes and/or incorporation of biorelevant cleavable links(e.g. reduction or acidic pH-sensitive bond) bridging the PFbackbone and pendant polymer brushes.8,9 Therefore theamphiphilic PF-backboned bb copolymers with hetero-polymerbrushes developed herein present a promising alternative forcancer theranostics.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors acknowledge the financial support from theNational Natural Science Foundation of China (Grants51473072 and 21504035), the Thousand Young TalentProgram, the Open Research Fund of State Key Laboratory ofPolymer Physics and Chemistry, Changchun Institute ofApplied Chemistry, Chinese Academy of Sciences, and theYoung Crop Plan of Hubei University of Traditional ChineseMedicine (2017ZZX021).
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